System and method for emulsion breaking

ABSTRACT

A method of recovering a bead support from an emulsion includes supplying an aqueous surfactant solution into a centrifuge tube; supplying a hydrophobic liquid over the surfactant solution in the centrifuge tube, wherein a ratio of the volume of the aqueous surfactant solution to the volume of the hydrophobic liquid is not greater than 0.5; and applying an emulsion over the hydrophobic liquid while centrifuging, the emulsion comprising a dispersed aqueous phase including the bead support, the emulsion breaking and material of the dispersed phase preferentially partitioning to the surfactant solution.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a divisional of U.S. application Ser. No. 15/375,271filed Dec. 12, 2016. U.S. application Ser. No. 15/375,271 is adivisional of U.S. application Ser. No. 14/536,429 filed Nov. 7, 2014(U.S. Pat. No. 9,533,240), which claims benefit of U.S. ProvisionalApplication No. 61/902,897 filed Nov. 12, 2013, and U.S. ProvisionalApplication No. 61/942,167 filed Feb. 20, 2014. All applications listedin this section are incorporated herein by reference, each in itsentirety.

FIELD OF THE DISCLOSURE

This disclosure, in general, relates to systems and methods for breakingemulsions.

BACKGROUND

Emulsions are utilized in a range of industries, including the food,biological sciences, and chemical industries. In particular, thechemical and biological sciences industries utilize emulsions to isolatevolumes of reactants. For example, the chemical industry utilizesemulsions for emulsion polymerization in which small volumes of monomersin solution are isolated as a dispersed phase and subsequentlypolymerized. In the biological sciences, particularly the geneticsciences, emulsions have been used to isolate genetic material intodispersed volumes, limiting cross-contamination between the dispersedvolumes. In a particular example, emulsions are utilized to isolatevolumes that include a bead support and a polynucleotide to beamplified. For example, a polynucleotide isolated in a small volume witha bead support, primers, a variety of nucleotides, enzymes, and othercofactors can be subjected to amplification conditions to facilitate theformation of nucleic acid beads incorporating the amplifiedpolynucleotide or complements thereof.

While the principles of amplification are understood, the process orautomation of this process has proven difficult. In particular,automation of the formation of the emulsion is challenging. Moreover,automating the recovery of the bead supports from an emulsion followingamplification has proven challenging. Often, a significant portion ofthe bead supports to which products of the amplified polynucleotide areattached are lost during the separation of the bead supports from theemulsion. As such, the yield of bead supports conjugated topolynucleotides is diminished. Such a loss of yield can adversely affectgenetic testing methods.

SUMMARY

In an example, a method of recovering a bead support from an emulsionincludes supplying an aqueous surfactant solution into a centrifugetube; supplying a hydrophobic liquid over the surfactant solution in thecentrifuge tube, wherein a ratio of the volume of the aqueous surfactantsolution to the volume of the hydrophobic liquid is not greater than0.5; and applying an emulsion over the hydrophobic liquid whilecentrifuging, the emulsion comprising a dispersed aqueous phaseincluding the bead support, the emulsion breaking and material of thedispersed phase preferentially partitioning to the surfactant solution.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure may be better understood, and its numerousfeatures and advantages made apparent to those skilled in the art byreferencing the accompanying drawings.

FIG. 1 includes a block flow diagram illustrating an exemplary methodfor recovering an amplified bead substrate.

FIG. 2 includes a block flow diagram illustrating an exemplary methodfor breaking an emulsion.

FIG. 3 includes an illustration of an exemplary centrifuge tube.

FIG. 4 includes an illustration of an exemplary centrifuge rotor.

FIG. 5 includes an illustration of an exemplary centrifuge tube.

FIG. 6 includes an illustration of an exemplary slinger or distributor.

FIG. 7, FIG. 8, FIG. 9, FIG. 10, and FIG. 11 include illustrations of anexemplary centrifuge device.

The use of the same reference symbols in different drawings indicatessimilar or identical items.

DETAILED DESCRIPTION

In an exemplary embodiment, a method of recovering a bead support froman emulsion includes applying an aqueous surfactant solution into acentrifuge tube. A hydrophobic liquid is applied over the surfactantsolution in the centrifuge tube. In an example, the ratio of the volumeof the aqueous surfactant solution to the volume of the hydrophobicliquid is not greater than 1.0, such is not greater than 0.5.Alternatively, the ratio of the initial volume of the aqueous surfactantsolution to the total volume of the centrifuge tube is not greater than0.5, such as not greater than 0.2 or not greater than 0.1. Thecentrifuge tube can be located within a centrifuge rotor. While rotatingthe centrifuge rotor including the centrifuge tube, an emulsion can beapplied over the hydrophobic liquid. The emulsion includes a dispersedaqueous phase including the bead support. As the emulsion breaks,material of the dispersed phase, including the bead support, canpreferentially partition into the surfactant solution. As such, the beadsupport is driven into the aqueous surfactant solution toward the bottomof the centrifuge tube in response to centrifugal force. The aqueoussurfactant solution can include a non-ionic surfactant or can include ananionic surfactant. In an example, the non-ionic surfactant includes apolyethylene glycol ether, an alkyl pyrrolidine, an alkylimidazolidinone, an alkyl morpholine, an alkyl imidazole, an alkylimidazoline, or a combination thereof. In another example, the non-ionicsurfactant includes a non-ionic fluorosurfactant. In a further example,the anionic surfactant includes a sulfate or sulfonate surfactant. Therecovered bead supports, when recovered as part of a genetic testingsystem, can be applied to a sequencing device and sequencing can beperformed.

For example, FIG. 1 includes an illustration of an exemplary method 100in which an emulsion is generated, as illustrated at 102. The emulsionincludes a bead support and a target polynucleotide in a dispersedphase. For example, reactants and cofactors along with a bead supportand a target polynucleotide can be isolated in small volumes of aqueoussolution as the dispersed phase.

The bead support can be formed of a polymer. An exemplary polymerincludes acrylamide, vinyl acetate, hydroxyalkylmethacrylate, apolyethylene glycol, derivations thereof or any combination thereof. Inan example, the bead support includes functional sites to which otherspecies, such as an oligonucleotide primer, click chemistry, or abinding agent, such as biotin or streptavidin, can be attached. Inparticular, the polymer of the bead support can be formed with anAcrydite™ comonomer having an acrylate modified with an oligonucleotide.In another example, the polymer bead support can include an amine orhydroxyl group that can be reactive with additional agents, such ashalides, amines, azide, cyanuric acid, di-isocyanate, bis-NHS esters, orany combination thereof, attached, for example, to a protein oroligonucleotide.

In the emulsion, an aqueous phase is distributed in a hydrophobic phase.In an example, the hydrophobic phase can include fluorinated liquids,minerals oils, silicone oils, or any combination thereof. Optionally,the hydrophobic phase can include a surfactant, such as a non-ionicsurfactant, such as a non-ionic surfactant described below.

The emulsion can be subjected to amplification conditions and as aresult, the target polynucleotide can be amplified and the products ofsuch an amplification can attached to the bead support, as illustratedat 104, forming a template bead support. For example, the targetpolynucleotide can be amplified using a polymerase chain reaction (PCR).In another example, the target polynucleotide can be amplified usingrecombinase polymerase amplification (RPA), such as isothermal RPA.

Following amplification, the emulsion can be broken, as illustrated at106, and the templated bead support washed. For example, the emulsioncan be applied continuously, such as in a stream, to a centrifuge tubedisposed on a rotating rotor of a centrifuge. The centrifuge tube caninclude an aqueous surfactant solution and optionally, the hydrophobicliquid disposed over the aqueous surfactant solution. When the emulsionis applied to the hydrophobic liquid, the dispersed phase is driven tothe interface between the hydrophobic liquid and the surfactantsolution. At the interface, the emulsion can break, driving the beadsupport associated with the dispersed phase into the surfactant solutionand toward the bottom of the centrifuge tube.

In a particular example, hydrophobic liquid includes fluorinatedliquids, minerals oils, silicone oils, or any combination thereof. Thehydrophobic liquid can further include a surfactant, such as a non-ionicsurfactant. Exemplary non-ionic surfactants are described below. Inparticular, the hydrophobic liquid is miscible with the continuous phaseof the emulsion. For example, the hydrophobic liquid can have the sameor similar composition to that of the continuous phase of the emulsion.

In particular, the emulsion is broken at an interface between thesurfactant solution and the hydrophobic liquid. As such, it has beenfound that the relative volumes and in particular, the associateddistance that the dispersed phase of the emulsion travels as it isapplied over the hydrophobic liquid influences the yield of beadsupports from the emulsion.

For example, a method 200 for breaking emulsion is illustrated in FIG.2. The method 200 includes providing an aqueous surfactant solution in acentrifuge tube, as illustrated at 202. In a particular example, theinitial volume of aqueous solution is in a range of 50 μL to 1 mL, suchas a range of 50 μL to 500 μL, a range of 50 μL to 250 μL, or even arange of 50 μL to 150 μL.

In an example, the surfactant solution can include one or moresurfactants having a total concentration in the range of 0.01% to 20% byweight. For example, surfactant can be included in a total amount in arange of 0.1% to 15.0%, such as a range of 0.5% to 10.0%, a range of0.5% to 5.0% or even a range of 0.5% to 3% by weight. In anotherexample, surfactant can be included in a total amount in a range of 5.0%to 20.0%, such as a range of 10.0% to 20.0%, or a range of 12.0% to18.0%.

The surfactant can be an ionic surfactant, an amphoteric surfactant, ora non-ionic surfactant. The ionic surfactant can be an anionicsurfactant. An exemplary anionic surfactant includes a sulfatesurfactant, a sulfonate surfactant, a phosphate surfactant, acarboxylate surfactant, or any combination thereof. An exemplary sulfatesurfactant includes alkyl sulfates, such as ammonium lauryl sulfate,sodium lauryl sulfate (sodium dodecyl sulfate, (SDS)), or a combinationthereof; an alkyl ether sulfate, such as sodium laureth sulfate, sodiummyreth sulfate, or any combination thereof; or any combination thereof.An exemplary sulfonate surfactant includes an alkyl sulfonate, such assodium dodecyl sulfonate; docusates such as dioctyl sodiumsulfosuccinate; alkyl benzyl sulfonate (e.g., dodecyl benzene sulfonicacid or salts thereof); or any combination thereof. An exemplaryphosphate surfactant includes alkyl aryl ether phosphate, alkyl etherphosphate, or any combination thereof. An exemplary carboxylic acidsurfactant includes alkyl carboxylates, such as fatty acid salts orsodium stearate; sodium lauroyl sarcosinate; a bile acid salt, such assodium deoxycholate; or any combination thereof.

In another example, the ionic surfactant can be a cationic surfactant.An exemplary cationic surfactant includes primary, secondary or tertiaryamines, quaternary ammonium surfactants, or any combination thereof. Anexemplary quaternary ammonium surfactant includes alkyltrimethylammoniumsalts, such as cetyl trimethylammonium bromide (CTAB) or cetyltrimethylammonium chloride (CTAC); cetylpyridinium chloride (CPC);polyethoxylated tallow amine (POEA); benzalkonium chloride (BAC);benzethonium chloride (BZT); 5-bromo-5-nitro-1,3-dioxane;dimethyldioctadecylammonium chloride; dioctadecyldimethylammoniumbromide (DODAB); or any combination thereof.

An exemplary amphoteric surfactant includes a primary, secondary, ortertiary amine or a quaternary ammonium cation with a sulfonate,carboxylate, or phosphate anion. An exemplary sulfonate amphotericsurfactant includes(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); a sultainesuch as cocamidopropyl hydroxysultaine; or any combination thereof. Anexemplary carboxylic acid amphoteric surfactant includes amino acids,imino acids, betaines such as cocamidopropyl betaine, or any combinationthereof. An exemplary phosphate amphoteric surfactant includes lecithin.

In another example, the surfactant can be a non-ionic surfactant such asa polyethylene glycol-based surfactant, an alkyl pyrrolidine surfactant,an alkyl imidazolidinone surfactant, an alkyl morpholine surfactant, analkyl imidazole surfactant, an alkyl imidazoline surfactant, or acombination thereof. In a particular example, thepolyethylene-glycol-based surfactant includes a polyethylene-glycolether, such as an alkylphenol polyethoxylate. In another example, thenon-ionic surfactant includes a non-ionic fluorosurfactant, such as anethoxylated fluorocarbon. In a further example, the surfactant solutioncan include octyl pyrrolidine.

In particular, the surfactant solution can include combinations of suchsurfactants. For example, the surfactant solution can include acombination of a non-ionic surfactant with an anionic surfactant. In aparticular example, the surfactant solution can include a non-ionicsurfactant, such as a polyethylene glycol ether, an alkyl pyrrolidine,or a non-ionic fluorosurfactant, and an anionic surfactant, such as asulfate surfactant, for example SDS. In particular, the surfactantsolution can include an ionic surfactant, such as an anionic surfactant,in an amount in a range of 0.1% to 20.0%, such as a range of 1.0% to15.0%, or a range of 5.0% to 15.0%, or a range of 8.0% to 12.0%. Inaddition, the surfactant solution can include a non-ionic surfactant,such as alkyl pyrrolidine (e.g., octyl pyrrolidine), in a range of 0.01%to 10.0%, such as a range of 0.05% to 8.0%, or a range of 1.0% to 6.0%.In another example, the surfactant solution can include a non-ionicsurfactant in a range of 0.05% to 3.0%.

Optionally, a hydrophobic liquid can be applied as a capping layer overthe surfactant solution, as illustrated at 204 in FIG. 2. Optionally,the hydrophobic liquid can be applied to the surfactant solution whilethe centrifuge is rotating. For example, the hydrophobic liquid caninclude an oil, such as a natural oil or a synthetic oil. Exemplary oilsinclude fluorinated liquids, minerals oils, silicone oils, or anycombination thereof, or any other suitable oil. In an example, thehydrophobic liquid can include a surfactant, such as a non-ionicsurfactant described above. In another example, the hydrophobic liquidis miscible with the continuous phase of the emulsion. In particular,the hydrophobic liquid can have the same or similar composition to thecontinuous phase of the emulsion.

In an example, the initial volume of the hydrophobic liquid is in arange of 100 μL to 10 mL, such as a range of 200 μL to 5 mL, a range of300 μL to 2.7 mL, or even a range of 500 μL to 2 mL. The initial ratioof the volume of the surfactant solution relative to the volume of thehydrophobic liquid applied over the surfactant solution is not greaterthan 1.0. For example, the ratio can be not greater than 0.5, such asnot greater than 0.25, not greater than 0.1, or even not greater than0.05. In general, the ratio is at least 0.01. As a result of the volumedifference, the relative heights of the solutions within the centrifugetube have a ratio of approximately that of the ratio of the volume.Alternatively, the initial volume of the hydrophobic liquid can beprovided during centrifugation or as part of dispensing the emulsion.

In an example, the centrifuge tube can have a total volume in a range of0.7 mL to 5 mL, such as a range of 0.8 mL to 3 mL, a range of 1.0 mL to2.5 mL, or even a range of 1.5 mL to 2.5 mL. The initial ratio of thevolume of the surfactant solution relative to the volume of thecentrifuge tube is not greater than 1.0. For example, the ratio can benot greater than 0.5, such as not greater than 0.25, not greater than0.1, or even not greater than 0.05. In general, the ratio is at least0.01.

For example, as illustrated by the centrifuge tube 302 of FIG. 3, thesurfactant solution 306 underlies the hydrophobic liquid forming layer308. The surfactant solution 306 has a height 310 and the hydrophobicliquid forms a layer 308 having a second height 312. As illustrated, theheight 312 of the hydrophobic liquid can be the same or greater than theheight 310 of the surfactant solution. The centrifuge tube 302 can alsoinclude a channel 304 through which additional material such as portionsof the hydrophobic liquid or the continuous phase of the emulsion can bedriven from the centrifuge tube 302 as the emulsion is applied to thecentrifuge tube and the aqueous dispersed phase is driven towards theinterface 314 between the hydrophobic liquid 308 and the surfactantsolution 306.

For example, a ratio of the initial height of the surfactant solution tothe initial height of the hydrophobic liquid is not greater than 1.0,such as not greater than 0.5, not greater than 0.25, not greater than0.1, or even not greater than 0.05. In particular, the ratio of heightscan be at least 0.01. Initially, the cumulative volume of the aqueoussurfactant solution and the hydrophobic liquid occupy a significantvolume of the centrifuge tube. For example, the cumulative volume canoccupy at least 80% of the tube volume, such as at least 90% of the tubevolume, at least 95% of the tube volume, or even 100% of the tubevolume.

As illustrated at 206 in FIG. 2, the emulsion is applied to thehydrophobic capping layer. In particular, the emulsion can be appliedwhile the centrifuge is rotating. When applied, the dispersed phase ofthe emulsion travels through the hydrophobic layer and concentrates. Thedispersed phase is driven into the surfactant solution. The beadsupports within the dispersed phase are released into the surfactantsolution and are driven to the bottom of the centrifuge tube. As theemulsion is applied, the relative volumes of the aqueous phase and thehydrophobic phase can change.

After the emulsion is broken, the bead supports are dispersed within thesolution. The aqueous solution can be washed, as illustrated at 208 inFIG. 2. For example, additional surfactant solution can be applied overthe bead supports to further remove oil and other contaminants. The washsolution can further include a sodium salt.

In an example, a significant portion of the bead supports are recoveredfollowing emulsion breaking. For example, at least 70% of the beadsupports in the emulsion can be recovered in the surfactant solution. Inparticular, at least 80% of the bead supports can be recovered, such asat least 85%, at least 90%, or even at least 95%, but not greater than100% of the bead supports can be recovered.

In a particular example in which the bead supports act as a support foran amplification product for use in a genetic testing system, adispersion including the bead support conjugated to amplificationproduct can be applied to an array of wells, as illustrated in FIG. 2 at210. The array of wells can form part of a device for use in a geneticsequencing system. As such, genetic testing can be performed on thepolynucleotides conjugated to the bead supports, as illustrated at 212.

Such a method is particularly useful in an automated system for breakingemulsion, in particular, those incorporated in an automated system forrecovering bead supports conjugated to an amplification product. In anexample, FIG. 4, FIG. 7, FIG. 8, FIG. 9, FIG. 10, and FIG. 11 illustratea centrifuge subsystem 700. A lid can be in the open configuration suchthat the interior of the centrifuge 700 can be seen as well as the tophousing rim on which the lid rests while in a closed configuration. Arotor 832 having a rotor housing 836 is visible through top housingaperture 840 defined by top housing room 828. Centrifuge 700 provides ahousing basin that is visible and defined by the top housing room 828.Housing basin 844 provides basin sidewalls 848 as well as a receivingplatform 852. Receiving platform 852 comprises motor mounting apertures856 as well as a central rotor axle aperture 860.

The rotor 832 and its rotor basin 864 comprise rotor basin sidewalls 868and rotor basin floor 870 and a rotor top rim 872 around the peripheryand upper end of rotor basin sidewalls 868. Rotor basin sidewalls 868provide at least one collection tube receptacle. The rotor basinsidewalls 868 provide first tube receptacle 876 and second tubereceptacle 880. Any number of tube receptacles can be provided in rotorbasin sidewall depending in part on the size of the tubes to be insertedand the overall size and surface area of the rotor basin sidewall 868.Rotor top rim 872 has an inner perimeter 873 and outer perimeter 874.Alongside the respective tube receptacles and provided in rotor top rim872 are collection tube exit channels receptacles. As illustrated inFIG. 4, first tube exit channel receptacle 884 and second tube exitchannel receptacle 888 are provided respectively with first and secondreceptacle grooves 886, 890. At the center of the rotor basin 864 is afluid distribution device or slinger receptacle 892 comprising slingerreceptacle sidewalls 894.

A fluid collection tube is provided comprising a main tube body and atube extension. The main tube body can comprise a main body sidewallsurrounding a tube interior comprising a tube opening at a first end,and a second end distal to the first end providing a sealed base. Thetube extension can comprise a tube extension sidewall defining a fluidexit channel in fluid communication with the tube interior through atube channel inlet proximal to the tube opening and extending to a tubechannel outlet distal to the tube opening. The fluid collection tube canfurther comprise a tube lip disposed about the perimeter of the tubeopening and allowing fluid communication of the tube exit channel withthe tube interior. An optional tube buttress disposed between the tubeextension sidewall and the main tube sidewall provides further supportand rigidity. The tube extension sidewall and the main tube sidewall canbe positioned at any angle relative to one another, from about 15° toabout 90°, from about 1.0° to about 80°, from about 5.0° to about 75°,from about 10° to about 65°, from about 20° to about 60°, from about 25°to about 50°, from about 30° to about 45°, from about 30° to about 60°,from about 40° to about 50°, or greater than about 90° relative to eachother.

The fluid collection tube can be provided in any suitable shapeincluding the main tube body and the tube extension. For example, themain tube body can comprise a generally cylindrical portion proximal thefirst end, a conically tapered portion proximal the second end, and arounded second end. For example, the tube exit channel and tubeextension sidewall can have a U-shaped cross-section along alongitudinal axis. The average cross-sectional area of the exit tubechannel can be less than, equal to, or greater than the averagecross-sectional area of the tube interior. The average cross-sectionalarea of the exit tube channel can be less than about 95%, less thanabout 90%, less than about 75%, less than about 60%, less than about50%, less than about 40%, less than about 25%, less than about 20%, lessthan about 15%, less than about 10%, less than about 5%, or less thanabout 1%, but at least 0.001% of the average cross-sectional area of thetube interior.

The fluid collection tube can optionally comprise a cap or lid. The lidcan be completely separable from the tube housing or can be permanentlyjoined to the housing, for example, through a flexible hinge. The capcan be configured to close off the main tube interior, the fluid exitchannel, or both.

The fluid collection tube can be constructed from any suitable material.Suitable materials include metals, plastics, glass, ceramics, or anycombination thereof. Examples of suitable plastics includepolypropylene, polycarbonate, and polyvinyl chloride. The fluidcollection tube can be constructed to contain any desired volume. Thefluid collection tube can have a volume of less than about 1 μL, fromabout 1 μL to about 1 L, from about 10 μL to about 1 dL, from about 100μL to about 1 cL, from about 500 μL to about 50 mL, from about 1 mL toabout 25 mL, from about 2.5 mL to about 15 mL, or from about 5 mL toabout 10 mL.

FIG. 5 illustrates a centrifuge tube or collection tube 896. Thecollection tube 896 comprises a tube housing 900 including a main tubebody 904, comprising a tube interior 902, and a tube extension 906comprising a fluid exit channel 908. The length of the collection tubeis defined by a first tube end 912 and a second tube end 916. At firsttube end 912, is a tube lip 920 that can be engaged by rotor sidewallapertures tube receptacles 876, 880. Fluid exit channel 908 has a lengthdefined by a tube channel inlet 924 and a tube channel outlet 928.

In particular, an angle 932 is shown defined by the spacing of the sidewalls 910 of tube extension 906 from main tube body 904. An optionaltube buttress can be formed between the main tube body and the tubeextension. Tube inlet 920 defines and surrounds a tube opening(aperture) 936 having a tube opening center 938. At the middle of tubeopening 936 is tube opening center 938.

A fluid distribution device 940, also referred to herein as a “slinger,”is illustrated in FIG. 6. The slinger can comprise sidewalls defining acentral channel comprising a first end, a central zone, and a second endalong a longitudinal axis. Sidewall lateral extensions also referred toas “wings” herein, can be provided, extending away from the sidewalls oneither side of the central channel. The wings are useful in mating withand ensuring a secure connection with a fluid distribution devicereceptacle of a centrifuge rotor. The slinger can have any number ofspouts that can be correlated with the number of fluid collection tubesto be used in conjunction with the slinger. For example, when two fluidcollection tubes are employed, the slinger can include a first spoutextending from the central zone to the first end and terminating at afirst spout outlet, the sidewalls tapering along the central zone towardthe first spout; and a second spout extending from the central zone tothe second end and terminating at a second spout outlet, the sidewallstapering along the central zone toward the second spout.

The slinger can be configured to mate with a fluid distribution devicereceptacle of a centrifuge rotor using any suitable configuration. Forexample, wings, as described herein, can be employed. Rather than use aninsertable/detachable slinger, a slinger can be employed that ispermanently or integrally associated with the rotor housing.

The slinger can be fabricated from any suitable material or materialssuch as those described herein for the collection tube. The slinger cancomprise one or more of the materials described herein for the fluidcollection tubes. The slinger can have any desired volume. The slingercan have a volume or less than about 1 μL, from about 1 μL to about 1 L,from about 10 μL to about 1 dL, from about 100 μL to about 1 cL, fromabout 500 μL to about 50 mL, from about 1 mL to about 25 mL, from about2.5 mL to about 15 mL, or from about 5 mL to about 10 mL.

FIG. 6 is a plan view of a liquid distribution device or slinger 940.Slinger 940 comprises a slinger housing 944, which is shaped to provideslinger sidewalls 948, a slinger base 952, and slinger wings 956. Aslinger central channel 960 is defined by the slinger base 952 andslinger sidewalls 948 running the length of the slinger. At the centerof the slinger is a central zone 962. While the slinger 940 is shownhaving a single central channel 960, additional central channels can beprovided in other embodiments. First slinger spout 964 and secondslinger spout 968 comprise opposite sides and ends of the slingercentral channel 960. At either end of slinger central channel 960 aswell as the ends of respective first slinger spouts 964, 968 are firstslinger spout outlet 972 and second slinger spout outlet 976.

A centrifuge rotor, which is particularly suitable for separatingwater-in-oil emulsions and removing the oil phase of such an emulsion.The centrifuge rotor can comprise a rotor housing having a bisectingrotor axis perpendicular to a central rotor axis, a rotor basin formedby the rotor housing, a rotor basin floor, and a rotor basin sidewalllining the rotor housing basin and extending up toward a rotor top rimhaving an inner perimeter and an outer perimeter. The rotor can furthercomprise at least one collection tube receptacle comprising an openingformed in the basin sidewall, and at least one tube extension receptaclehaving a grove formed in the rotor top rim and extending from the innerperimeter to the outer perimeter. The sidewalls can comprise asubstantially flat inset region about the collection tube receptacle andadjacent the tube receptacle opening.

The centrifuge rotor can be provided with any number of collection tubereceptacles and corresponding tube extensions. Generally, an even numberof receptacles are provided on opposite sides of the rotor basin. Forexample, first and second tube receptacles positioned opposite eachother along the bisecting rotor axis; and first and second tubeextension grooves opposite each other along the bisecting rotor axis. Anodd number of receptacles can be utilized, and in such embodiments therotor can be balanced to account for the weight of any unpairedreceptacles. Balancing can also be provided for embodiments where aneven number of receptacles are provided but when not all receptacles arefitted with collection tubes or collection tubes of unequal volume orweight. The rotor can also comprise at least one liquid distributiondevice (slinger) receptacle extending from the rotor basin floor andhaving a distribution device receptacle longitudinal axis.

The collection tube receptacle opening and the tube extension groove aregenerally positioned at an angle relative to each other corresponding tothe angle of the tube extension sidewall relative to the main tube body.For example, the collect tube receptacle and the tube extension groovecan be from about 15° to about 90°, from about 1.0° to about 80°, fromabout 5.0° to about 75°, from about 10° to about 65°, from about 20° toabout 60°, from about 25° to about 50°, from about 30° to about 45°,from about 30° to about 60°, from about 40° to about 50°, or greaterthan about 90° relative to each other. The shape of the rotor sidewallcan be configured to accommodate a collection tube buttress. The fluiddistribution device receptacle can include a means for receiving orreversibly locking in place the slinger. For example, the slingerreceptacle can contain opposing sidewalls on either side of thedistribution device receptacle longitudinal axis that can cooperate withwings on the slinger.

The slinger receptacle can be provided in any suitable configurationwithin the centrifuge rotor. The bisecting rotor axis can be parallel ornon-parallel to the distribution device receptacle longitudinal axis.The axes can be positioned from about 0.01° to about 25°, from about0.05° to about 20°, from about 0.15° to about 15.0°, from about 0.25° toabout 10.0°, from about 0.5° to about 5.0°, from about 1.0° to about2.5°, less than about 0.01°, or greater than about 25° relative to eachother.

FIG. 7 is atop perspective view of rotor 832 as positioned in housingbasin 844. First collection tube 896 and second collection tube 898 aredisposed in respective first and second tube receptacles 876 and 880.Slinger 940 is positioned in slinger receptacle 892. Collection tubes896 and 898 each have respective tube openings 936 and 980, which inturn have respective tube opening centers 938 and 882. Cutting the rotorapproximately in half is a bisecting rotor axis 984, which can beperpendicular to central rotor axis 986, extending into and out of thepage. Along the slinger central channel 960, along the length of slinger940 and extending out either end is a slinger longitudinal axis 988.Slinger 940 is mounted in slinger receptacle 892 so that slinger 940 isoffset from the centers of the respective collection tubes 896, 898.Rather than the slinger spout outlet 972 being directly in line with thefirst tube opening center 938, there is an offset of and by an angle 992defined by the spacing of bisecting rotor axis 984 from slingerlongitudinal axis 988. Angle 992 can be varied as appropriate. Theoffset of slinger 940 from the respective tube opening centers accountsfor angular movement of the rotor such that when in motion any fluidexiting respective first slinger spout outlets 972, 976 will land at ornear respective tube opening centers 938, 932. The offset angle 992 canbe varied to account for changes in the angular speed of or accelerationfor the rotor 832.

When the centrifuge is run in accordance with the present teachings,fluid from the fluid collection tubes is expelled from the rotor throughthe tube exit channels. The expelled fluid can be accumulated,processed, or discarded using any suitable means or mechanism. Forexample a peripheral gutter can be employed. The peripheral gutter caninclude a gutter housing providing a top gutter surface, a bottom guttersurface, and gutter sidewalls extending between the top and bottomgutter surfaces along an outer gutter perimeter of the gutter housing. Agutter inlet can be located along an inner perimeter of the gutterhousing. The gutter can be connected to the centrifuge housing using anymeans or mechanism. For example, a gutter flange extending around thegutter outer perimeter and adapted for placement on the top housing rimcan be employed. The fluid collected in the peripheral gutter can bediscarded using any means or mechanism. For example, a gutter outlet canbe provided in the gutter housing along with a housing drainage aperturein the housing sidewall and a basin drainage aperture in the housingbasin sidewall. In such a configuration, drainage tubing can beoperatively associated with the gutter outlet and pass through thedrainage apertures.

FIG. 8 shows a top perspective view of centrifuge subsystem 700 asassembled with various components including peripheral gutter 996, firstand second collection tubes 896, 898, and slinger 940. A drainage tubing1036 is shown engaged with gutter outlet 1024 and can be passed throughbasin drainage aperture 782 and housing drain aperture 780. A sideperspective view of centrifuge subsystem 700 as assembled is also shownin FIG. 9.

A cross-sectional view of the assembled centrifuge subsystem 700 isshown in FIG. 10. Rotor 832 rests on and is connected to rotor axle 824of motor 816. First and second collection tubes 896, 898 are shownfilled with fluid comprising an oil/air interface 1040 separating an oilphase 1044 the emulsion. Collection tubes 896,898 can comprise at leastone mixing ball each in addition aqueous solution. Such bead or beadsare located at the base or second tube end 916. When the rotor is inangular momentum the oil phase can exit the collection tubes through therespective exit tube channels and land in peripheral gutter 996. Oilcollected in the volume 1016 of the gutter 996 can drain through port1024.

When in the centrifuge, the surfactant solution 1048 is disposed withthe volume of the centrifuge tube and resides proximal to the lower ordistal end of the centrifuge tube. Once hydrophobic liquid 1802 isapplied over the surfactant solution 1048, a liquids interface 1804 isformed. The hydrophobic liquid forms an upper interface 1040. When theemulsion hits the upper interface 1040, the dispersed phase of theemulsion is driven through the hydrophobic liquid 1802 to the liquidsinterface 1804. The aqueous phase of the emulsion enters the surfactantsolution 1048. As the continuous phase of the emulsion fills thecentrifuge tube, the level of hydrophobic liquid increases and excessliquid is driven out of the collection tubes and into the gutter 996.

The centrifuge of the present teachings is particular suitable for thebreaking of emulsions. The emulsion can be in form a sample, i.e.,source fluid. Any means or mechanism can be employed to deliver sourcefluid to the centrifuge. The sample fluid can be delivered through aninlet supply line (fluid supply line). When a lid is not employed, or isnot in a closed configuration, the fluid line can be delivered directlyinto the centrifuge through the top centrifuge opening afforded by thecentrifuge housing sidewalls. When a lid is employed and is in a closedconfiguration, the fluid supply line can pass through at least one of alid aperture and a housing aperture to gain access to the interior ofthe centrifuge. For example, the lid can comprise a housing with a toplid surface and a bottom lid surface, a lid aperture extending from thetop lid surface to the bottom lid surface. The lid aperture can belocated anywhere on the lid, for example, it can be centrally located.More than one lid aperture can be provided. The lid aperture can beconfigured to accept a fluid supply line and positioned above the fluiddistribution device when the lid is in a closed configuration. A fluidsupply source can be in fluid communication with the fluid supply line.A fluid supply pump can be configured to pump fluid from the fluidsupply source, through the fluid supply line and into the centrifuge.

Centrifuge subsystem 700 is shown in FIG. 11 in a closed configurationand operably connected to a fluid source. A lid aperture adapter 1052 isconnected to centrifuge subsystem 700 through central lid aperture 744.Inlet fluid line 1056 can pass either directly into centrifuge subsystem700 or through one or more of a lid aperture adapter and a fluid lineconnector 1060. Fluid line 1056 can originate from a fluid sample source1070. Fluid sample source 1070 can be supplied to centrifuge subsystem700 by means of a fluid sample source pump 1064. As shown in FIG. 11,pump 1064 is a syringe style pump. The present teachings allow for useof other types of pumps as well or in addition to a syringe style pump.For example, a peristaltic pump or diaphragm pump could be employed aspump 1064. Fluid line 1056 can originate from an emulsion subsystem 300.The sample fluid supplied by inlet fluid 1056 can comprise awater-in-oil emulsion. The water phase of the water and oil emulsion cancomprise microreactors that in turn can contain PCR product or products.

The above system and method of recovering a bead support from anemulsion provides particular technical advantages over other recoveryprocesses. In particular, the above methods improve recovery of beadsupports, expressed as a percent recovery.

In a first aspect, a method of recovering a bead support from anemulsion includes supplying an aqueous surfactant solution into acentrifuge tube; supplying a hydrophobic liquid over the surfactantsolution in the centrifuge tube, wherein a ratio of the volume of theaqueous surfactant solution to the volume of the hydrophobic liquid isnot greater than 0.5; and applying an emulsion over the hydrophobicliquid while centrifuging, the emulsion comprising a dispersed aqueousphase including the bead support, the emulsion breaking and material ofthe dispersed phase preferentially partitioning to the surfactantsolution.

In an example of the first aspect, the ratio is not greater than 0.25.For example, the ratio is not greater than 0.1, such as not greater than0.05. In particular, the ratio is at least 0.01.

In another example of the first aspect and the above examples, supplyingthe hydrophobic liquid includes supplying the hydrophobic liquid whilecentrifuging.

In a further example of the first aspect and the above examples, themethod further includes preparing the emulsion including the dispersedphase including the bead support. For example, the method furtherincludes amplifying a target polynucleotide to generate amplifiedpolynucleotide on the bead support. In an example, amplifying includeperforming polymerase chain reaction. In another example, amplifyinginclude performing RPA.

In an additional example of the first aspect and the above examples, themethod further includes washing the separated bead support. For example,the method further includes applying the separated bead support to asequencing device.

In another example of the first aspect and the above examples, thesurfactant solution includes a surfactant in an amount in a range of0.1% to 10%.

In a further example of the first aspect and the above examples, thesurfactant solution includes a nonionic surfactant.

In an additional example of the first aspect and the above examples, thenonionic surfactant includes a polyethylene glycol ether.

In another example of the first aspect and the above examples, thesurfactant solution includes an anionic surfactant. For example, theanionic surfactant includes a sulfonate surfactant. In another example,the sulfonate surfactant includes dodecyl benzene sulfonic acid or asalt thereof.

In a further example of the first aspect and the above examples, thesurfactant solution includes a fluorosurfactant. For example, thefluorosurfactant is an ethoxylated non-ionic fluorosurfactant.

In a second aspect, a method of recovering a bead support from anemulsion includes supplying an aqueous surfactant solution into acentrifuge tube; supplying a hydrophobic liquid over the surfactantsolution in the centrifuge tube, wherein a ratio of the volume of theaqueous surfactant solution to the volume of the hydrophobic liquid isnot greater than 0.5; and applying an emulsion over the hydrophobicliquid while centrifuging, the emulsion comprising a dispersed aqueousphase including the bead support, the emulsion breaking and material ofthe dispersed phase preferentially partitioning to the surfactantsolution.

In an example of the second aspect, the ratio is not greater than 0.25,such as not greater than 0.1, or not greater than 0.05, and at least0.01.

In another example of the second aspect and the above example, supplyingthe hydrophobic liquid includes supplying the hydrophobic liquid whilecentrifuging.

In a further example of the second aspect and the above example, themethod further includes preparing the emulsion including the dispersedphase including the bead support. For example, the method furtherincludes amplifying a target polynucleotide to generate amplifiedpolynucleotide on the bead support. In another example, amplifyinginclude performing polymerase chain reaction. In an additional example,amplifying include performing recombinase polymerase amplification.

In an additional example of the second aspect and the above example, themethod further includes washing the separated bead support. For example,the method further includes applying the separated bead support to asequencing device.

In another example of the second aspect and the above example, thesurfactant solution includes surfactant in a total amount in a range of0.01% to 20.0%.

In a further example of the second aspect and the above example, thesurfactant solution includes a nonionic surfactant. For example, thenonionic surfactant is selected from the group consisting of apolyethylene glycol-based surfactant, an alkyl pyrrolidine surfactant,an alkyl imidazolidinone surfactant, an alkyl morpholine surfactant, analkyl imidazole surfactant, an alkyl imidazoline surfactant, and acombination thereof. In a particular example, the nonionic surfactantincludes an alkyl pyrrolidine.

In an additional example of the second aspect and the above example, thesurfactant solution includes an anionic surfactant. For example, theanionic surfactant includes a sulfonate surfactant. For example, thesulfonate surfactant include dodecyl benzene sulfonic acid or a saltthereof.

In a further example of the second aspect and the above example, thesurfactant solution includes an anionic surfactant in a range of 5.0% to15.0% and includes a non-ionic surfactant in a range of 0.05% to 8.0%.

In a third aspect, a method of recovering a bead support from anemulsion includes supplying an aqueous surfactant solution into acentrifuge tube, the surfactant solution includes an anionic surfactantin a range of 5.0% to 15.0% and includes a non-ionic surfactant in arange of 0.05% to 8.0%; and applying an emulsion into the centrifugetube while centrifuging, the emulsion comprising a dispersed aqueousphase including the bead support and a continuous phase comprising ahydrophobic liquid, the emulsion breaking and material of the dispersedphase preferentially partitioning to the surfactant solution.

In an example of the third aspect, a ratio of the volume of the aqueoussurfactant solution to the volume of the centrifuge tube is not greaterthan 0.5. For example, the ratio is not greater than 0.25, not greaterthan 0.1 or not greater than 0.05 and at least 0.01.

In another example of the third aspect and the above example, the methodfurther includes preparing the emulsion including the dispersed phaseincluding the bead support. In an additional example, the method furtherincludes amplifying a target polynucleotide to generate amplifiedpolynucleotide on the bead support.

In a further example of the third aspect and the above example, themethod further includes washing the separated bead support. For example,the method further includes applying the separated bead support to asequencing device. In an example, the nonionic surfactant is selectedfrom the group consisting of a polyethylene glycol-based surfactant, analkyl pyrrolidine surfactant, an alkyl imidazolidinone surfactant, analkyl morpholine surfactant, an alkyl imidazole surfactant, an alkylimidazoline surfactant, and a combination thereof. For example, thenonionic surfactant includes an alkyl pyrrolidine.

In an additional example of the third aspect and the above example, theanionic surfactant includes a sulfonate surfactant. For example, thesulfonate surfactant include dodecyl benzene sulfonic acid or a saltthereof.

Note that not all of the activities described above in the generaldescription or the examples are required, that a portion of a specificactivity may not be required, and that one or more further activitiesmay be performed in addition to those described. Still further, theorder in which activities are listed are not necessarily the order inwhich they are performed.

In the foregoing specification, the concepts have been described withreference to specific embodiments. However, one of ordinary skill in theart appreciates that various modifications and changes can be madewithout departing from the scope of the invention as set forth in theclaims below. Accordingly, the specification and figures are to beregarded in an illustrative rather than a restrictive sense, and allsuch modifications are intended to be included within the scope ofinvention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the use of “a” or “an” are employed to describe elements andcomponents described herein. This is done merely for convenience and togive a general sense of the scope of the invention. This descriptionshould be read to include one or at least one and the singular alsoincludes the plural unless it is obvious that it is meant otherwise.

Benefits, other advantages, and solutions to problems have beendescribed above with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any feature(s) that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as a critical, required, or essentialfeature of any or all the claims.

After reading the specification, skilled artisans will appreciate thatcertain features are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, references to valuesstated in ranges include each and every value within that range.

What is claimed:
 1. A method of recovering a bead support from anemulsion, the method comprising: supplying an aqueous surfactantsolution into a centrifuge tube; the surfactant solution including ananionic surfactant in a range of 5.0% to 15.0% and including a non-ionicsurfactant in a range of 0.05% to 8.0%, wherein a ratio of the volume ofthe aqueous surfactant solution to the volume of the centrifuge tube isnot greater than 0.5 and at least 0.01; and preparing an emulsioncomprising a sample of template bead supports in a dispersed aqueousphase and a hydrophobic liquid in a continuous phase; applying theemulsion into the centrifuge tube while centrifuging; recovering thesample of template bead supports from the surfactant solution; washingthe recovered sample of template bead supports; and applying the washedsample of template bead supports to an array of wells, wherein the arrayof wells forms a part of a genetic sequencing system for performinggenetic testing on the sample.
 2. The method of claim 1, wherein thenonionic surfactant is selected from a polyethylene glycol-basedsurfactant, an alkyl pyrrolidine surfactant, an alkyl imidazolidinonesurfactant, an alkyl morpholine surfactant, an alkyl imidazolesurfactant, an alkyl imidazoline surfactant, and a combination thereof.3. The method of claim 2, wherein the nonionic surfactant includes octylpyrrolidine.
 4. The method of claim 1, wherein the anionic surfactantincludes a sulfonate surfactant.
 5. The method of claim 4, wherein thesulfonate surfactant includes dodecyl benzene sulfonic acid or a saltthereof.
 6. The method of claim 1, further comprising applying a cappinglayer comprising a hydrophobic liquid into the centrifuge tube over theaqueous surfactant solution.
 7. The method of claim 6, wherein thehydrophobic liquid is selected from a fluorinated liquid, a natural oil,a synthetic oil, and combinations thereof.